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Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 2 of 23The text below applies only to use of the data by the United States EnvironmentalProtection Agency (US EPA) in connection with the provisions of the FederalInsecticide, Fungicide, and Rodenticide Act (FIFRA).Statement of No Data Confidentiality ClaimNo claim of confidentiality is made for any information contained in this study on thebasis of its falling within the scope of FIFRA § 10(d)(l)(A), (B), or (C).We submit this material to the United States Environmental Protection Agencyspecifically under the requirements set forth in FIFRA as amended, and consent to theuse and disclosure of this material by EPA strictly in accordance with FIFRA. Bysubmitting this material to EPA in accordance with the method and format requirementscontained in PR Notice 86-5, we reserve and do not waive any rights involving thismaterial that are or can be claimed by the company notwithstanding this submission toEPA.Company:Monsanto CompanyCompany Agent: ___________________________________________________Title:Signature:_____________________________________________________________________________________ Date:____________
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 3of 23Statement of ComplianceThis study meets the US EPA Good Laboratory Practice requirements as specified in40 CFR Part 160.Submitter: __________________ Date:________SponsorRepresentative: XK.Date: ~Study Director: __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Date: / 2 Ž~95
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 4of 23Study Title:Study Number: 05-01-62-02Quality Assurance StatementAssessment of the in vitro <strong>Digestibility</strong> of the CrylA. 105 Proteinin Simulated Gastric FluidReviews conducted by the Quality Assurance Unit confirm that the final report accuratelydescribes the methods and standard operating procedures followed and accurately reflectsthe raw data of the study.Following is a list of reviews conducted by the Monsanto Regulatory Quality AssuranceUnit on the study reported herein.Dates of Inspection /PhaseDate Reported To:Audit Study Director Management08/04/2005 Digestive Fate 08/04/2005 08/04/200507/27/2005 Western Blot 08/04/2005 08/04/200510/19/2005 Raw Data Audit 10/25/2005 10/25/200510/19/2005 Draft Report Audit 10/27/2005 10/27/2005(Qality ASSurancd UnitMonsanto Regulatory, Monsanto Companydate
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 5 of 23Study Number: 05-01-62-02Study Information PageTitle: Assessment of the in vitro <strong>Digestibility</strong> of the CrylIA. 105Protein in Simulated Gastric FluidPrimary Testing Facility:Study Director:Principal Investigator:Monsanto CompanyProduct Characterization Center800 North Lindbergh BoulevardSt. Louis, MO 63167Elena A. Rice, Ph.D.Monsanto CompanyProduct Characterization Center800 North Lindbergh BoulevardSt. Louis, Missouri 63167Shefalee A. KapadiaMonsanto CompanyProduct Characterization Center800 North Lindbergh BoulevardSt. Louis, Missouri 63167Study Initiation Date: July 15, 2005Study Completion Date: November 2, 2005Records Retention:Specimen Retention:All study specific raw data, protocol, amendments,deviations, final report, and facility records will be retainedat Monsanto, St. Louis.Specimens will be retained at Monsanto, St. Louis, asspecified in section 5.2.© 2005 Monsanto Company. All Rights Reserved.This document is protected under copyright law. This document is for use only by theregulatory authority to which this has been submitted by Monsanto Company, and only insupport of actions requested by Monsanto Company. Any other use of this material,without prior written consent of Monsanto, is strictly prohibited. By submitting thisdocument, Monsanto does not grant any party or entity any right or license to theinformnation or intellectual property described in this document.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 6of 23Study Certification PageThe results reported in this Final Report accurately reflect the data generated under studynumber 05-01-62-02.Approved BY:Elena A. Rice, Ph.D.Study DirectorMonsanto CompanyProduct Characterization CenterDate'_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 0)~~~~~~~~- 06ary A~Bannon, Ph.D.Lead, Protein Sciences TeamMonsanto CompanyProduct Characterization CenterDate
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 7 of 23Table of ContentsTitle......................................................................................................................................1Statement of No Data Confidentiality Claim.......................................................................2Statement of Compliance.....................................................................................................3Quality Assurance Statement...............................................................................................4Study Information Page .......................................................................................................5Study Certification Page ......................................................................................................6Table of Contents.................................................................................................................7Abbreviations.......................................................................................................................91.0 Summary................................................................................................................102.0 Introduction............................................................................................................103.0 Purpose...................................................................................................................114.0 Materials ................................................................................................................114.1 Test Substance ...........................................................................................114.2 Control Substance ......................................................................................114.3 Reference Substance ..................................................................................114.4. Characterization of the Test Substance......................................................115.0 Test System............................................................................................................115.1 Justification for Selection of the Test System............................................125.2 Specimens ..................................................................................................125.3 Procedure for Identification of Specimens ................................................136.0 Experimental Design..............................................................................................136.1 <strong>Digestibility</strong> of the Test Substance in SGF................................................136.2 Experimental Controls ...............................................................................147.0 Analytical Methods................................................................................................147.1 SGF Activity Assay ...................................................................................147.2 SDS-PAGE ................................................................................................157.3 Colloidal Brilliant Blue G Staining............................................................167.4 Western Blot Analysis ...............................................................................167.5 Statistical Methods.....................................................................................178.0 Control of Bias.......................................................................................................17
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 8 of 239.0 Rejected Data.........................................................................................................1710.0 Protocol Amendments............................................................................................1811.0 Protocol Deviations................................................................................................1812.0 Results and Discussion ..........................................................................................1812.1 <strong>Pepsin</strong> Activity in SGF ..............................................................................1812.2 Assessment of the Extent of Digestion of the <strong>Cry1A.105</strong> Protein by <strong>Pepsin</strong>using Colloidal Brilliant Blue G Gel Staining of SDS-PAGE...................1812.3 Assessment of the Extent of Digestion of the <strong>Cry1A.105</strong> Protein by <strong>Pepsin</strong>using Western Blot Analysis......................................................................1913.0 Conclusions............................................................................................................2014.0 References..............................................................................................................20FiguresFigure 1. Colloidal Brilliant Blue G stained SDS-polyacrylamide gels ...........................21Figure 2. Western blot analysis.........................................................................................22AppendixList of Applicable SOPs.........................................................................................23
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 9 of 23Abbreviations 1CEWCorn earwormCFRCode of Federal RegulationsDFDilution factorEC 50Effective protein concentration to inhibit the growth of thetarget insect by 50%ECLEnhanced chemiluminescenceE. coli Escherichia coliEPAEnvironmental Protection AgencyFIFRAFederal Insecticide, Fungicide, and Rodenticide ActHRPHorseradish peroxidaseIgGImmunoglobulin GLODLimit of DetectionILSIInternational Life Science InstituteLBLaemmli bufferMALDI-TOF MS Matrix assisted laser desorption ionization – time of flight massspectrometryNFDMNon-fat dry milkPBSTPhosphate buffered saline containing Tween-20SDS-PAGESodium dodecyl sulfate-polyacrylamide gel electrophoresisSGFSimulated gastric fluidSOPStandard operating procedureTTimeTCATrichloroacetic acidUSUnited States1× LB Laemmli buffer [62.5mM Tris-HCl, 5% (v/v) 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 0.005%(w/v) bromophenol blue, 10% (v/v) glycerol, pH 6.8].5× LB Five times concentrated 1× LB1 Standard abbreviations, e.g. units of measure, concentration, mass, time, etc., are used without definitionaccording to the format described in “Instructions to Authors” in The Journal of Biological Chemistry.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 10 of 231.0 SummaryMonsanto has developed plants that produce the <strong>Cry1A.105</strong> insecticidal protein and areprotected from feeding damage caused by European corn borer (Ostrinia nubilalis) andother lepidopteran insect pests. <strong>Cry1A.105</strong> is a modified Bacillus thuringiensis Cry1Aprotein with 93.6 % overall amino acid sequence identity to the Cry1Ac protein.The purpose of this study was to assess the in vitro digestibility of the <strong>Cry1A.105</strong> proteinin simulated gastric fluid (SGF) containing the proteolytic enzyme pepsin. The<strong>Cry1A.105</strong> protein used in this study was produced in and purified from E. coli. TheE. coli-produced <strong>Cry1A.105</strong> protein was characterized prior to the digestibility study.<strong>Digestibility</strong> of the <strong>Cry1A.105</strong> protein in SGF was assessed using stained SDSpolyacrylamidegel and western blot analysis.The results of this study demonstrated that the full-length <strong>Cry1A.105</strong> protein was rapidlydigested during incubation in SGF. At least 99.3% of the full-length <strong>Cry1A.105</strong> proteinwas digested within 30 seconds when analyzed using Colloidal Brilliant Blue G stainedpolyacrylamide gels. Greater than 95% of the <strong>Cry1A.105</strong> protein was digested in SGFwithin 30 seconds when specimens were analyzed using western blot. A faint ~4.5 kDaband was observed between the 30-second and 20-minute digestion time points whenanalyzed using a Colloidal Brilliant Blue G stained polyacrylamide gel. Neither thisband, nor any other immunoreactive band was detected using western blot analysis.2.0 IntroductionMonsanto has developed plants that produce the <strong>Cry1A.105</strong> insecticidal protein and areprotected from feeding damage caused by European corn borer (Ostrinia nubilalis) andother lepidopteran insect pests. <strong>Cry1A.105</strong> is a modified Bacillus thuringiensis Cry1Aprotein with 93.6 % overall amino acid sequence identity to the Cry1Ac protein.Currently, proteins introduced into commercial food crops through the techniques ofbiotechnology are evaluated for safety, including an assessment of the potential to beallergenic. One aspect of this assessment includes analysis of the digestibility of thetarget protein in an SGF assay containing pepsin. The correlation between proteinallergenicity and protein stability in an in vitro pepsin digestion assay has beenpreviously established (Astwood et al., 1996). Proteins that are highly digestible areexpected to be less likely to cause sensitization or allergenic reaction when consumed.Recently, the International Life Science Institute (ILSI) standardized the pepsindigestibility assay protocol in a multi-laboratory evaluation (Thomas et al., 2004). TheSGF formulation, time course, and experimental parameters used in this study followedthe conditions used in the ILSI multi-laboratory evaluation.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 11 of 233.0 PurposeThe purpose of this study was to assess the stability of the <strong>Cry1A.105</strong> protein insimulated gastric fluid containing the proteolytic enzyme pepsin.4.0 Materials4.1 Test SubstanceThe test substance was the <strong>Cry1A.105</strong> protein. The <strong>Cry1A.105</strong> protein(Analytical Protein Standard lot 20-100073) was isolated from a fermentationbatch of E. coli containing pMON96851 expression plasmid. This protein hasbeen characterized and has a total protein concentration of 1.2 mg/ml and a purityof 92 %. Functional activity was confirmed using an insect bioassay with thelarvae of a susceptible pest, corn earworm (CEW). The EC 50 value was 5.8 ng/mlof diet. Prior to its application to the test system, the test substance was stored ina –80 ºC freezer in a test substance storage buffer containing 25 mM CAPS,1 mM benzamidine-HCl, 0.1 mM EDTA, and 0.2 mM DTT, pH ~10.3.4.2 Control SubstanceThere was no control substance for this study.4.3 Reference SubstanceThere was no reference substance for this study. Analytical reference standards(e.g., molecular weight markers) used in this study were documented in the dataand are described in this report.4.4. Characterization of the Test SubstanceThe characterization of the physicochemical and functional properties of the testsubstance was performed under characterization plan 20-100073 and is describedon the Certificate of Analysis. The following properties were established for the<strong>Cry1A.105</strong> protein: identity (N-terminal sequencing, MALDI-TOF massspectrometry (MS), immunodetection), concentration (amino acid analysis),purity (SDS-PAGE/densitometry), molecular weight (SDS-PAGE/densitometry,MALDI-TOF MS), stability (SDS-PAGE/densitometry) and activity (CEWbioassay).5.0 Test SystemThe test system for this study was simulated gastric fluid (SGF) that contains theproteolytic enzyme pepsin. The SGF was prepared using a highly purified form of pepsin(Catalog number P-6887, Sigma Company, St. Louis, MO). The SGF was formulated sothat ten units of pepsin activity per microgram of total protein from the test substancewould be present in the digestion reactions. The amount of pepsin powder used to
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 12 of 23prepare SGF was calculated from the specific activity reported on the product label. Oneunit of activity is defined as a change in A 280 nm of 0.001 per minute at 37 °C, measuredas trichloroacetic acid (TCA) soluble products using hemoglobin as the substrate. Thestock SGF solution was prepared by adding pepsin powder (26.6 mg) to 33.2 ml of anacidic sodium chloride solution (2 mg/ml NaCl, 10 mM HCl, pH 1.3). After the activityof pepsin in SGF was confirmed, the stock SGF solution was diluted to provideapproximately 1500 units pepsin activity/ml of solution.5.1 Justification for Selection of the Test SystemIn vitro digestion models are used widely to assess the nutritional value ofingested proteins based on their amino acid bioavailability. Also, the correlationbetween protein allergenicity and protein stability in an in vitro pepsin digestionassay has been previously established (Astwood et al., 1996). Recently the pepsindigestibility assay protocol was standardized by ILSI in a multi-laboratoryevaluation test (Thomas et al., 2004). This multi-laboratory test showed thatresults of the in vitro pepsin digestion assay are reproducible when a commonprotocol is followed. The SGF formulation, time course, and experimentalparameters used in this study followed the conditions used in the ILSI multilaboratoryevaluation test.5.2 SpecimensSpecimens were generated by incubating the test substance with the test systemfor the times specified in section 5.3. See Sections 6.0 through 7.0 for details onthe preparation and analysis of specimens. Specimens will be retained in a -80 °Cfreezer one year, after which they will no longer afford analytical evaluation andmay be discarded.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 14 of 236.2 Experimental ControlsExperimental controls were prepared to determine the stability of the testsubstance in the test system buffer lacking pepsin [10 mM HCl, 2 mg/ml NaCl,pH 1.3]. These experimental controls were identified with the letter "P". Thezero incubation time point (P0) was prepared in a separate tube. Test systembuffer (80 µl) was quenched by addition of 35 µl of carbonate buffer and 35 µl of5× LB prior to the addition of 20 µl of the diluted test substance. The 60 minincubation time point (P7) was prepared by adding 20 µl of the diluted testsubstance to test system buffer lacking pepsin (80 µl). The tube was vortex mixedand immediately placed in a 37.1 ºC water bath. After 60 min of incubation, thesample was quenched by addition of 35 µl of carbonate buffer and 35 µl of 5× LB.Additional experimental controls were prepared to evaluate the stability of thepepsin in the test system (SGF) lacking the test substance and to determine if nonspecificinteraction occurs between the test system components and the antibodiesduring western blot analysis of the specimens. These experimental controlscontained an aliquot of the test system incubated with test substance storagebuffer instead of the test substance and were identified with the letter "N". Thezero incubation time point (N0) was in a separate tube. Test system (80 µl) wasquenched by addition of 35 µl of carbonate buffer and 35 µl of 5× LB prior toaddition of 20 µl of the storage buffer (25 mM CAPS, pH 10.3, 1 mMbenzamidine-HCl, 0.1 mM EDTA, and 0.2 mM DTT). The 60 min incubationtime point (N7) was prepared by adding 20 µl storage buffer to 80 µl of testsystem. The tube was vortex mixed and placed in a 37.1 ºC water bath. After60 min of incubation, the sample was quenched by addition of 35 µl of carbonatebuffer and 35 µl of 5× SB.All experimental controls were heated at 75-100 ºC for 5-10 min, frozen on dryice, and stored in a -80 ºC freezer until analyzed.7.0 Analytical MethodsActivity of the SGF was assessed using a pepsin activity assay. The digestibility of the<strong>Cry1A.105</strong> protein in SGF was assessed using stained polyacrylamide gels and westernblot analysis. The limit of detection (LOD) of the <strong>Cry1A.105</strong> protein for these methodswas determined concurrently.7.1 SGF Activity AssayThe SGF activity assay was used to confirm the suitability of the test systembefore its use with the test substance. Acceptable activity was defined as a pepsinactivity per mg of pepsin powder (0.03 mg of powder per ml of diluted SGF)equal to the activity of pepsin per mg of pepsin powder as determined by themanufacturer (±1000 units/mg). One unit of pepsin activity in this assay isdefined as the amount of pepsin that will produce a change in the absorbance at
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 15 of 23280 nm of 0.001 per min at pH 1.2-2.0 at 37 ± 2 ºC. The assay is used to estimatethe amount of soluble peptides present in a TCA solution after pepsin digestion ofdenatured hemoglobin. Undigested hemoglobin was precipitated with TCA, andthe amount of soluble peptides was estimated by measuring the absorbance at280 nm. The amount of soluble peptide is directly proportional to the amount ofprotease activity.Briefly, the SGF was diluted to 0.03 mg of solid material (pepsin) per ml of SGF[the dilution factor (DF) was 26.7]. Acidified hemoglobin [2% (w/v), 5 ml] wasadded to each of three replicates of the test sample and blank samples and prewarmedat 37 ± 2 °C for 5-10 minutes prior to starting the reactions. Diluted SGF(1 ml) was added to each replicate of test samples and both test and blank sampleswere incubated at 37.0 °C for an additional 10 min. The reaction was stopped byaddition of 10 ml of chilled 5% (v/v) TCA to the test and blank samples. DilutedSGF (1 ml) was then added to the blank samples. Samples were mixed and thenincubated another 5-10 min at 37.4 °C. Precipitated protein was removed byfiltering the test and the blank samples using 0.8 µm syringe filters. Samples ofthe clarified test and blank samples were read at 280 nm in a Beckman DU-650Spectrophotometer. The activity of pepsin was calculated using the followingequation:MeanTestA 280 nm− Mean BlankA280nm× DF ,0.001×10min×1mlwhere 0.001 is the change in the absorbance at 280 nm per min at pH 1.2-2.0 and37 ± 2 ºC produced by one unit of pepsin activity; 10 min is the reaction time,1 ml is the amount of SGF added to the reaction; and, DF is the dilution factor forthe SGF.7.2 SDS-PAGESamples containing 1× LB from the SGF in vitro digestion of the <strong>Cry1A.105</strong>protein were separated by SDS-PAGE using pre-cast tricine 10-20%polyacrylamide gradient mini-gels and tricine running buffer (Invitrogen,Carlsbad, CA). The protein loaded per lane was based on the pre-digestion totalprotein concentration of the <strong>Cry1A.105</strong> protein. All experimental controls wereloaded at the same volume as those containing <strong>Cry1A.105</strong> protein so that all othercomponents would be comparable. All samples were heated at 100.3 °C for5 min prior to loading on the gels. Protein markers were used to estimate therelative molecular weight. Electrophoresis was performed at a constant voltage of125 V for 85 minutes. After electrophoresis, proteins were either visualized bystaining the gel with colloidal Brilliant Blue G (section 7.3), or the gel wassubjected to electrotransfer of proteins to nitrocellulose membrane for westernblot analysis (section 7.4).
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 16 of 237.3 Colloidal Brilliant Blue G StainingThe colloidal Brilliant Blue G staining method was selected because it is aneffective method for detecting nanogram quantities of protein on a gel (Neuhoff etal., 1988). Mark12 molecular weight markers (Invitrogen, Carlsbad, CA) wereused to estimate the relative molecular weight of visualized proteins and peptides.Based on pre-digestion concentrations, approximately 0.7 µg of total protein wasloaded per lane. After separation of proteins, the gels were fixed in a solutioncontaining 7% (v/v) acetic acid and 40% (v/v) methanol for 30 min and stainedfor approximately 20 h in 1× Brilliant Blue G-colloidal stain solution containing20% (v/v) methanol. The gels were briefly destained for 30 s in a 10% (v/v)acetic acid, 25% (v/v) methanol solution and completely destained for ~5 h in a25% (v/v) methanol solution. Images were captured using a Bio-Rad GS-800densitometer. The results of the in vitro digestibility of <strong>Cry1A.105</strong> protein weredetermined by visual examination of the stained gels.The approximate molecular weights of the full-size protein and proteolyticfragment observed on the colloidal Brilliant Blue G stained gels were visuallydetermined relative to the positions of the molecular weight markers.The LOD of the <strong>Cry1A.105</strong> protein using the colloidal Brilliant Blue G stainingprocedure was determined. Various dilutions of the zero time point (T0)digestion specimen were loaded onto a separate gel that was run concurrently withthe gel used to assess digestibility. Aliquots of the T0 digestion samplerepresenting approximately 700, 350, 100, 50, 20, 10, 5, and 2.5 ng total proteinwere used for the stained LOD gel.7.4 Western Blot AnalysisSpecimens from the SGF in vitro digestions were also analyzed using westernblotting. Based on pre-digestion concentrations, approximately 20 ng of totalprotein were loaded per lane. Following electrophoresis, pre-stained molecularweight markers (Precision Plus Protein Standards, Bio-Rad, Hercules CA) wereused to verify electrotransfer of proteins to the membrane. Proteins wereelectrotransferred to nitrocellulose membranes (0.45 µm pore size, Invitrogen) for90 min at a constant voltage of 25 V.Membranes were blocked overnight in a 4 ºC refrigerator with 5% (w/v) non-fatdry milk (NFDM) in phosphate buffered saline containing Tween-20 (PBST)buffer. All subsequent incubations (described below) were performed at roomtemperature. Membranes were incubated with rabbit anti–<strong>Cry1A.105</strong> antibody(lot 070705JL) diluted 1:2,000 in PBST containing 1% (w/v) NFDM for 1 h.Excess serum was removed by three 10 min washes with PBST. The membranewas incubated with HRP–conjugated goat anti-rabbit IgG (Sigma) at a dilution of1:10,000 in PBST containing 1% (w/v) NFDM for 1 h and again washed
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 17 of 23(three 10 min washes) with PBST. Immunoreactive bands were visualized usingthe enhanced chemiluminescence (ECL) detection system (AmershamBiosciences) and exposed (2, 5, and 10 minutes) to Hyperfilm ECL highperformance chemiluminescence film (Amersham Biosciences). Films weredeveloped using a Konica SRX101A automated film processor (Tokyo, Japan).The approximate molecular weights of the full-size protein observed on thewestern blots were visually determined relative to the positions of the molecularweight markers.The LOD for the <strong>Cry1A.105</strong> protein using the western blot analysis procedurewas determined. Various dilutions of the zero time point (T0) digestion specimenwere loaded onto a separate gel that was run concurrently with the digestionwestern blot gel and subjected to the same western blot procedure as describedabove. Aliquots of the T0 digestion sample representing approximately 7, 3.5, 2,1, 0.5, 0.2, 0.1, and 0.05 ng total protein were used for the western blot LODanalysis.7.5 Statistical MethodsNo statistical analysis was performed.8.0 Control of BiasMeasures taken to control bias in this study were the inclusion of both stability and testsystem experimental controls to account for any effects due to the model in the absenceof the pepsin enzyme and the absence of the test substance. Digestion specimens andlower limit of detection samples were analyzed concurrently to eliminate run-to-runvariation.9.0 Rejected DataOne set of data, which included determination of pepsin activity, in vitro digestibility of<strong>Cry1A.105</strong> in SGF, and western blot analysis of <strong>Cry1A.105</strong> SGF digestions was rejectedbecause this set of data was generated before the protocol was amended to includedilution of the test substance and vortexing of the digestion tube throughout the durationof the digestion to improve exposure of the test protein to the test system. One set ofSDS-PAGE gels was rejected because the band representing pepsin in the T7 specimenwas not observed on the gel, which most probably was a result of a loading error. Oneset of western blots was rejected because a significant reduction in the amount of the fulllength<strong>Cry1A.105</strong> protein in the T0 specimen was observed. This was likely caused by aloading error.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 18 of 2310.0 Protocol AmendmentsThe protocol contained a few typographical errors, which were corrected by amendments.Section 6.1, describing digestibility of the test substance in SGF, was amended to addressthe possibility of test substance precipitation and/or aggregation at low pH. There was nonegative impact on the study as a result of these changes.11.0 Protocol DeviationsThroughout the experimental phase of the study, one protocol deviation occurred. Thepreparation of the T0 specimen and experimental controls P0, P7, N0, and N7 followedthe amended procedure for the digestibility of the test substance, even though they werenot specifically referenced in the amendment. This deviation improved the quality of thestudy data, because the T0 specimen and experimental controls were prepared in a similarmanner to the study specimens.12.0 Results and Discussion12.1 <strong>Pepsin</strong> Activity in SGFThe pepsin activity in SGF was evaluated before conducting the digestion trials toassess the suitability of the test system used in this study. The experimentallyobserved activity was 2429 units per mg pepsin powder, which was within theacceptable interval of pepsin activity (2280 to 4280 units per mg pepsin powder).Therefore, the test system was shown to be suitable for use in this study.12.2 Assessment of the Extent of Digestion of the <strong>Cry1A.105</strong> Protein by <strong>Pepsin</strong>using Colloidal Brilliant Blue G Gel Staining of SDS-PAGEThe extent of digestion of the <strong>Cry1A.105</strong> protein was evaluated by visual analysisof colloidal Brilliant Blue G stained polyacrylamide gels (Figure 1). The SDS-PAGE for the digestibility assessment (Figure 1A) was run concurrently with aseparate SDS-PAGE to determine the LOD of <strong>Cry1A.105</strong> protein (Figure 1B).The limit of detection of the full-length (~130 kDa) <strong>Cry1A.105</strong> protein wasvisually estimated to be 0.005 µg or approximately 1% of the total protein loaded:0.005µg × 100%≅ 0.7%0.7 µ gThe gel used to assess the stability of the <strong>Cry1A.105</strong> protein to pepsin(Figure 1A) was loaded with ~0.7 µg (based on pre-digestion concentrations) foreach of the digestion time points. Visual examination of the stained gel showedthat the full-length (~130 kDa) <strong>Cry1A.105</strong> protein was digested below LODwithin 30 seconds of digestion in SGF (Figure 1A, lane 5). Therefore, at least99.3% (100% – 0.7% = 99.3%) of the full-length <strong>Cry1A.105</strong> protein was digestedwithin 30 seconds of incubation of <strong>Cry1A.105</strong> protein in SGF based on theColloidal Brilliant Blue G Gel stained SDS-PAGE analysis. A faint band with amolecular weight of approximately 4.5 kDa was observed at a very low level
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 19 of 23between the 30-second and 20-minute digestion time points (Figure 1A, lanes 5-9). No protein band was visible at the 30-minute digestion time point (Figure 1A,lane 10).No change in the full-length <strong>Cry1A.105</strong> protein band intensity was observed inthe absence of pepsin in the experimental controls P0 and P7 (Figure 1A, lanes 3and 12). This indicates that digestion of the <strong>Cry1A.105</strong> protein was due to theproteolytic activity of pepsin present in SGF and not due to the instability of thetest substance at pH 1.3 and 37°C.The experimental controls evaluating the stability of the pepsin in the test system(SGF) lacking the test substance demonstrated that pepsin was observed as thestained protein band at ~38 kDa throughout the experimental phase (Figure 1A,lanes 2 and 13). The amount of pepsin slightly decreased between 30 and 60 minof the digestion, most probably due to enzyme auto-digestion.12.3 Assessment of the Extent of Digestion of the <strong>Cry1A.105</strong> Protein by <strong>Pepsin</strong>using Western Blot AnalysisThe extent of digestion of the <strong>Cry1A.105</strong> protein was also evaluated by a westernblot method (Figure 2). The western blot used to assess the stability of the<strong>Cry1A.105</strong> protein to pepsin digestion (Figure 2A) was run concurrently with awestern blot to determine the LOD of <strong>Cry1A.105</strong> protein (Figure 2B). The LODof full-length (~130 kDa) <strong>Cry1A.105</strong> protein was visually estimated to be 1 ng or5 % of the total protein loaded:1ng× 100%= 5%20ngThe gel used to assess the <strong>Cry1A.105</strong> protein in vitro digestibility by western blotwas loaded with 20 ng total protein of the test substance (based on pre-digestionconcentrations) for each of the digestion time points. Western blot analysisdemonstrated that the <strong>Cry1A.105</strong> protein was digested below the LOD within30 seconds of incubation in SGF (Figure 2A, lane 5). Based on the western blotLOD for the <strong>Cry1A.105</strong> protein in SGF and the observation that no full-lengthprotein or immunoreactive bands were observed on the western blot at the 30-second digestion time point, it was concluded that at least 95%( 100 % − 5% = 95%) of the full-length <strong>Cry1A.105</strong> protein was digested within30 seconds.No change in the full-length <strong>Cry1A.105</strong> protein band intensity was observed inthe absence of pepsin in the experimental controls P0 and P7 (Figure 2A, lanes 3and 12). This indicates that the test substance was stable in the test systemwithout pepsin at pH 1.3 and ~37 ºC over the course of the experiment.No immunoreactive bands were observed in specimens N0 and N7 that representtest system experimental controls (Figure 2A, lanes 2 and 13). This indicates that
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 20 of 23non-specific interactions between the test system components and the antibodieswere not observed under these experimental conditions.13.0 ConclusionsThe results of this study demonstrated that the full-length <strong>Cry1A.105</strong> protein was rapidlydigested after incubation in SGF. At least 99.3% of the full-length <strong>Cry1A.105</strong> proteinwas digested within 30 seconds when analyzed using Colloidal Brilliant Blue G stainedpolyacrylamide gels. Greater than 95% of the <strong>Cry1A.105</strong> protein was digested in SGFwithin 30 seconds when specimens were analyzed using western blot analysis. A faint~4.5 kDa band was observed between the 30-second and 20-minute digestion time pointswhen analyzed using Colloidal Brilliant Blue G stained polyacrylamide gels. Neither thisband, nor any other immunoreactive band was detected when using western blot analysis.14.0 ReferencesAstwood, J. D., Leach, J. N., and Fuchs, R. L. (1996). Stability of food allergens todigestion in vitro. Nat. Biotechnol. 14, 1269-1273.Neuhoff, V., Norbert, A., Taube, D., and Wolfgang, E. (1988). Improved staining ofproteins in polyacrylamide gels including isoelectric focusing gels with clearbackground at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255-262.Thomas, K., Aalbers, M., Bannon, G. A., Bartels, M., Dearman, R. J., Esdaile, D. J., Fu,T. J., Glatt, C. M., Hadfield, N., Hatzos, C., Hefle, S. L., Heylings, J. R.,Goodman, R. E., Henry, B., Herouet, C., Holsapple, M., Ladics, G. S., Landry, T.D., MacIntosh, S. C., Rice, E. A., Privalle, L. S., Steiner, H. Y., Teshima, R., VanRee, R., Woolhiser, M., and Zawodny, J. (2004). A multi-laboratory evaluation ofa common in vitro pepsin digestion assay protocol used in assessing the safety ofnovel proteins. Regul. Toxicol. Pharmacol. 39, 87-98.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 21 of 23AFull-length 200<strong>Cry1A.105</strong>116.397.466.355.4<strong>Pepsin</strong>36.53121.514.463.52.51 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10B200116.397.466.355.436.53121.514.463.52.5Lane Sample Incubation Lane Sample AmountTime (min)(µg)1 Molecular weight marker — 1 Molecular weight marker —2 N0, SGF only 0 2 T0, protein+SGF 0.73 P0, protein only 0 3 T0, protein+SGF 0.354 T0, protein+SGF 0 4 T0, protein+SGF 0.15 T1, protein+SGF 0.5 5 T0, protein+SGF 0.056 T2, protein+SGF 2 6 T0, protein+SGF 0.027 T3, protein+SGF 5 7 T0, protein+SGF 0.018 T4, protein+SGF 10 8 T0, protein+SGF 0.0059 T5, protein+SGF 20 9 T0, protein+SGF 0.002510 T6, protein+SGF 30 10 Molecular weight marker —11 T7, protein+SGF 6012 P7, protein only 6013 N7, SGF only 6014 Molecular weight marker —Figure 1. Colloidal Brilliant Blue G stained SDS-polyacrylamide gelsPanel A corresponds to <strong>Cry1A.105</strong> protein digestion in SGF. Based on pre-digestionprotein concentrations, ~0.7 µg (total protein) was loaded in lanes containing <strong>Cry1A.105</strong>protein. The incubation times are indicated. Panel B corresponds to the limit ofdetection of <strong>Cry1A.105</strong> protein. Approximate molecular weights (kDa) are shown on theleft and correspond to the markers loaded in each gel. In both gels, <strong>Cry1A.105</strong> proteinmigrated to approximately 130 kDa and pepsin to approximately 38 kDa (indicated bythe arrows on the left). Blank or empty lanes were cropped and lanes renumbered.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 22 of 23Full-length<strong>Cry1A.105</strong>A25015010075503725201510B1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 1025015010075503725201510Lane Sample Incubation Lane Sample AmountTime (min)(ng)1 Molecular weight marker — 1 Molecular weight marker —2 N0, SGF only 0 2 T0, protein+SGF 73 P0, protein only 0 3 T0, protein+SGF 3.54 T0, protein+SGF 0 4 T0, protein+SGF 25 T1, protein+SGF 0.5 5 T0, protein+SGF 16 T2, protein+SGF 2 6 T0, protein+SGF 0.57 T3, protein+SGF 5 7 T0, protein+SGF 0.28 T4, protein+SGF 10 8 T0, protein+SGF 0.19 T5, protein+SGF 20 9 T0, protein+SGF 0.0510 T6, protein+SGF 30 10 Molecular weight marker —11 T7, protein+SGF 6012 P7, protein only 6013 N7, SGF only 6014 Molecular weight marker —Figure 2. Western blot analysisPanel A corresponds to <strong>Cry1A.105</strong> protein digestion in SGF. Based on pre-digestionprotein concentrations, 20 ng (total protein) was loaded in lanes containing <strong>Cry1A.105</strong>protein. The incubation times are indicated. Panel B corresponds to the limit ofdetection of the <strong>Cry1A.105</strong> protein. Approximate molecular weights (kDa) are shown onthe left and correspond to the markers loaded in each gel. In both gels, <strong>Cry1A.105</strong>migrated to approximately 130 kDa. A 10 min exposure is shown. Blank or empty laneswere cropped and lanes renumbered.
Monsanto Company Study: 05-01-62-02Final Report MSL: 19929Product Characterization Center Page 23 of 23Appendix. List of Applicable SOPsBR-ME-0388-02BR-ME-0392-01BR-ME-0460-02BR-ME-0527-01BR-ME-0924-01BR-ME-0973-01BR-EQ-0599-02BR-EQ-0857-01Sodium Dodecyl Sulfate Polyacrylamide Gel ElectrophoresisWestern Blot Analysis (Immunoblotting)Assay for <strong>Pepsin</strong> Activity in Simulated Gastric FluidBrilliant Blue G-Colloidal Staining of Polyacrylamide GelsElectrotransfer of Proteins to MembranesDrying of Polyacrylamide Mini Gels Using Invitrogen Gel DryingSystem (Adaptation of Invitrogen Gel Drying Procedure)Bio-Rad GS-710 and GS-800 DensitometersBeckman Coulter DU-650 Spectrophotometer